Characterization of Transcriptional Repressor Gene MSX1 Variations for Possible Associations with Congenital Heart Diseases

Background

The human heart consists of several cell types with distinct lineage origins. Interactions between these cardiac progenitors are very important for heart formation. The muscle segment homeobox gene family plays a key role in the cell morphogenesis and growth, controlled cellular proliferation, differentiation, and apoptosis, but the relationships between the genetic abnormalities and CHD phenotypes still remain largely unknown. The aim of this work was to evaluate variations in MSX1 and MSX2 for their possible associations with CHD.

Methods

We sequenced the MSX1 and MSX2 genes for 300 Chinese Han CHD patients and 400 normal controls and identified the variations. The statistical analyses were conducted using Chi-Square Tests as implemented in SPSS (version 19.0). The Hardy-Weinberg equilibrium test of the population was carried out using the online software OEGE.

Results

Six variations rs4647952, rs2048152, rs4242182, rs61739543, rs111542301 and rs3087539 were identified in the MSX2 gene, but the genetic heterozygosity of those SNPs was very low. In contrast, the genetic heterozygosity of two variations rs3821949 near the 5’UTR and rs12532 within 3’UTR of the MSX1 gene was considerably high. Statistical analyses showed that rs3821949 and rs12532 were associated with the risk of CHD (specifically VSD).

Conclusions

The SNPs rs3821949 and rs12532 in the MSX1 gene were associated with CHD in Chinese Han populations.     

A three-step protocol for lead optimization: quick identification of key conformational features and functional groups in the SAR studies of non-ATP competitive MK2 (MAPKAPK2) inhibitors

A three-step protocol for SAR development was introduced and applied to the SAR studies of the MK2 inhibitor program. Following this protocol, key conformational features and functional groups for improving MK2 inhibitor activity were quickly identified. Through this effort, the initial gap observed between in vitro binding activity and cellular activity in the lead identification stage was very much reduced. Compound 28 was identified with single digit binding activity (IC(50)=8 nM) and good cellular activity (EC(50)=310 nM). This provides further evidence that non-ATP-competitive binding MK2 inhibitors are feasible by targeting the outside ATP pocket.     

A 40-bp insertion/deletion polymorphism in the constitutive promoter of MDM2 confers risk for hepatocellular carcinoma in a Chinese population

The pathogenesis of HCC is a multistage process with the involvement of genetic factors. The aim of the present study is to investigate the possible association between a 40-bp insertion/deletion polymorphism (indel) at constitutive promoter of MDM2 and risk of hepatocellular carcinoma (HCC) in a Chinese population. Using 420 HCC patients and 423 control subjects, we genotyped the indel polymorphism (rs3730485) using polymerase chain reaction method. Logistic regression was used to analyze the association between the polymorphism and HCC susceptibility. Under co-dominant model, we found that the ins/del and del/del genotype of indel was associated with a significantly increased risk of HCC compared with its homozygote ins/ins (OR=1.39, 95%C.I.=1.03-1.87; OR=1.68, 95%C.I.=1.03-2.73, respectively). Presence of 40-bp deletion allele of MDM2 seemed to confer higher risk for HCC when compared with non-carriers (OR=1.30, 95%C.I.=1.06-1.60, P=0.011). Further stratification analysis showed that this association was more pronounced in patients with a family history of HCC, early tumor stage and higher serum alpha-fetoprotein (AFP). These findings indicated that the MDM2 indel polymorphism may be a genetic modifier for developing HCC in Chinese population.     

A chimeric human-cat fusion protein blocks cat-induced allergy

Animal allergens are an important cause of asthma and allergic rhinitis. We designed and tested a chimeric human-cat fusion protein composed of a truncated human IgG Fcgamma1 and the major cat allergen Fel d1, as a proof of concept for a new approach to allergy immunotherapy. This Fcgamma-Fel d1 protein induced dose-dependent inhibition of Fel d1-driven IgE-mediated histamine release from cat-allergic donors' basophils and sensitized human cord blood-derived mast cells. Such inhibition was associated with altered Syk and ERK signaling. The Fcgamma-Fel d1 protein also blocked in vivo reactivity in FcepsilonRIalpha transgenic mice passively sensitized with human IgE antibody to cat and in Balb/c mice actively sensitized against Fel d1. The Fcgamma-Fel d1 protein alone did not induce mediator release. Chimeric human Fcgamma-allergen fusion proteins may provide a new therapeutic platform for the immune-based therapy of allergic disease.     

Syntheses of quinolone hydrochloride enantiomers from synthons (R)- and (S)-2-methylpiperazine

A series of R and S enantiomers of 7-(3-methylpiperazin-1-yl) quinolone derivatives were synthesized from (R)- and (S)-tert-butyl 2-methylpiperazine-1-carboxylate and tested for their antibacterial activities on 14 kinds of bacteria. Although no distinct difference in in vitro antibacterial activities was observed, 2-64-fold difference between R and S enantiomers was observed in approximately 52% of cases.     

Development of a microelectronic chip array for high-throughput genotyping of Helicobacter species and screening for antimicrobial resistance

A microelectronic array assay was developed to specifically genotype Helicobacter pylori versus Helicobacter heilmannii and to determine antimicrobial resistance. Helicobacter 16S rRNA and 23S rRNA genes were specifically generated with Helicobacter genus-specific primers, respectively. The single-nucleotide polymorphisms (SNPs) in 16S rRNA, 268T specific in the H. pylori sequence, and 263A specific in H. heilmannii were used as molecular markers for identification of H. pylori and H. heilmannii, respectively. A triple-base-pair resistant mutation, AGA965-967TTC in 16S rRNA, is known to be responsible for H. pylori tetracycline resistance and was detected to identify resistant strains. H. pylori macrolide resistance was determined by the identification of 3 defined mutations in the 23S rRNA gene using the same method. The assay could be directly used to detect H. pylori in feces. The assay performs multiple determinations, including identification of Helicobacter species and antibiotic resistances, on the same microelectronic platform and is highly amenable to the development of other DNA-based assays.     

Role of dual-site phospholamban phosphorylation in intermittent hypoxia-induced cardioprotection against ischemia-reperfusion injury

Cardioprotection by intermittent high-altitude (IHA) hypoxia against ischemia-reperfusion (I/R) injury is associated with Ca(2+) overload reduction. Phospholamban (PLB) phosphorylation relieves cardiac sarcoplasmic reticulum (SR) Ca(2+)-pump ATPase, a critical regulator in intracellular Ca(2+) cycling, from inhibition. To test the hypothesis that IHA hypoxia increases PLB phosphorylation and that such an effect plays a role in cardioprotection, we compared the time-dependent changes in the PLB phosphorylation at Ser(16) (PKA site) and Thr(17) (CaMKII site) in perfused normoxic rat hearts with those in IHA hypoxic rat hearts submitted to 30-min ischemia (I30) followed by 30-min reperfusion (R30). IHA hypoxia improved postischemic contractile recovery, reduced the maximum extent of ischemic contracture, and attenuated I/R-induced depression in Ca(2+)-pump ATPase activity. Although the PLB protein levels remained constant during I/R in both groups, Ser(16) phosphorylation increased at I30 and 1 min of reperfusion (R1) but decreased at R30 in normoxic hearts. IHA hypoxia upregulated the increase further at I30 and R1. Thr(17) phosphorylation decreased at I30, R1, and R30 in normoxic hearts, but IHA hypoxia attenuated the depression at R1 and R30. Moreover, PKA inhibitor H89 abolished IHA hypoxia-induced increase in Ser(16) phosphorylation, Ca(2+)-pump ATPase activity, and the recovery of cardiac performance after ischemia. CaMKII inhibitor KN-93 also abolished the beneficial effects of IHA hypoxia on Thr(17) phosphorylation, Ca(2+)-pump ATPase activity, and the postischemic contractile recovery. These findings indicate that IHA hypoxia mitigates I/R-induced depression in SR Ca(2+)-pump ATPase activity by upregulating dual-site PLB phosphorylation, which may consequently contribute to IHA hypoxia-induced cardioprotection against I/R injury.     

Reassessment of suitable markers for taxonomy of Chaetophorales (Chlorophyceae,Chlorophyta) based on chloroplast genomes

Filamentous green algae Chaetophorales present numerous taxonomic problems as many other green algae. Phylogenetic analyses based on nuclear genes have limited solutions. Studies with appropriate chloroplast molecular markers may solve this problems; however, suitable molecular markers for the order Chaetophorales are still unknown. In this study, 50 chloroplast genomes of Chlorophyceae, including 15 of Chaetophorales, were subjected to single protein-coding gene phylogenetic analyses, and substitution rate and evolutionary rate assays, and PCR amplification verification was conducted to screen the suitable molecular markers. Phylogenetic analyses of three chloroplast representative genes (psaB, tufA, and rbcL) amplified from 124 strains of Chaetophorales showed that phylogenetic relationships were not improved by increasing the number of samples, implying that the genes themselves, rather than limited samples, were the reason for the unsupported Topology I. Seven genes (atpF, atpI, ccsA, cemA, chlB, psbB, and rpl2) with robust support were selected to be the most suitable molecular markers for phylogenetic analyses of Chaetophorales, and the concatenated seven genes could replace the time-consuming and labor-intensive phylogenetic analyses based on chloroplast genome to some extent. To further solve the taxonomic problems of Chaetophorales, suitable chloroplast markers combined with more taxon-rich approach could be helpful and efficient.